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Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Luchini A, Espina V, Liotta LA - Nat Commun (2014)

Bottom Line: The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface.We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP.We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling.

View Article: PubMed Central - PubMed

Affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.

ABSTRACT
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

No MeSH data available.


Related in: MedlinePlus

Molecular paints rapidly coat native proteins in solution.(a–c) Association (blue) and dissociation (red) binding curves (moles of paint per mole of protein) for paint molecules (structures are shown inset) associating with CA II. Molecular paints have unusually high association rates and low dissociation rates: saturation is reached within 5 min and the off rate is <10% dissociation after 2 h. (d) Calibration data for RBB associated to CA (absorbance spectra for RBB, CA and the complex is reported in Supplementary Fig. 7). (e) Scatchard plot of CA and AO50 shows the number of binding sites to be five, confirming the saturation point of the binding kinetics in (b). (f) Scatchard plot of CA and ANSA shows the number of binding sites to be three.
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f2: Molecular paints rapidly coat native proteins in solution.(a–c) Association (blue) and dissociation (red) binding curves (moles of paint per mole of protein) for paint molecules (structures are shown inset) associating with CA II. Molecular paints have unusually high association rates and low dissociation rates: saturation is reached within 5 min and the off rate is <10% dissociation after 2 h. (d) Calibration data for RBB associated to CA (absorbance spectra for RBB, CA and the complex is reported in Supplementary Fig. 7). (e) Scatchard plot of CA and AO50 shows the number of binding sites to be five, confirming the saturation point of the binding kinetics in (b). (f) Scatchard plot of CA and ANSA shows the number of binding sites to be three.

Mentions: We identified a panel of small, synthetic aryl hydrocarbon containing organic dyes (Fig. 1; Supplementary Figs 1 and 2; Supplementary Table 1), from a large number of candidate molecules (Supplementary Table 2), that bind to proteins as molecular paints. Paint chemistries were selected because they have extremely rapid on-rates (units: M−1 s−1) and very slow off-rates (<10−5 s−1, Supplementary Table 1) that are 10–100 times higher than most protein–protein interactions1920 (Fig. 2, using at least 10-fold molar excess of dye paint will coat 83% of low-affinity transient interactions that have been characterized21, Supplementary Fig. 3). Paint chemistries remain bound following protein dissociation or denaturation with 2 M urea (Fig. 3; Supplementary Table 1), and bind to multiple sites on the exposed protein surface to achieve complete masking of all the trypsin cleavage sites (Fig. 4; Supplementary Fig. 4).


Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Luchini A, Espina V, Liotta LA - Nat Commun (2014)

Molecular paints rapidly coat native proteins in solution.(a–c) Association (blue) and dissociation (red) binding curves (moles of paint per mole of protein) for paint molecules (structures are shown inset) associating with CA II. Molecular paints have unusually high association rates and low dissociation rates: saturation is reached within 5 min and the off rate is <10% dissociation after 2 h. (d) Calibration data for RBB associated to CA (absorbance spectra for RBB, CA and the complex is reported in Supplementary Fig. 7). (e) Scatchard plot of CA and AO50 shows the number of binding sites to be five, confirming the saturation point of the binding kinetics in (b). (f) Scatchard plot of CA and ANSA shows the number of binding sites to be three.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109009&req=5

f2: Molecular paints rapidly coat native proteins in solution.(a–c) Association (blue) and dissociation (red) binding curves (moles of paint per mole of protein) for paint molecules (structures are shown inset) associating with CA II. Molecular paints have unusually high association rates and low dissociation rates: saturation is reached within 5 min and the off rate is <10% dissociation after 2 h. (d) Calibration data for RBB associated to CA (absorbance spectra for RBB, CA and the complex is reported in Supplementary Fig. 7). (e) Scatchard plot of CA and AO50 shows the number of binding sites to be five, confirming the saturation point of the binding kinetics in (b). (f) Scatchard plot of CA and ANSA shows the number of binding sites to be three.
Mentions: We identified a panel of small, synthetic aryl hydrocarbon containing organic dyes (Fig. 1; Supplementary Figs 1 and 2; Supplementary Table 1), from a large number of candidate molecules (Supplementary Table 2), that bind to proteins as molecular paints. Paint chemistries were selected because they have extremely rapid on-rates (units: M−1 s−1) and very slow off-rates (<10−5 s−1, Supplementary Table 1) that are 10–100 times higher than most protein–protein interactions1920 (Fig. 2, using at least 10-fold molar excess of dye paint will coat 83% of low-affinity transient interactions that have been characterized21, Supplementary Fig. 3). Paint chemistries remain bound following protein dissociation or denaturation with 2 M urea (Fig. 3; Supplementary Table 1), and bind to multiple sites on the exposed protein surface to achieve complete masking of all the trypsin cleavage sites (Fig. 4; Supplementary Fig. 4).

Bottom Line: The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface.We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP.We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling.

View Article: PubMed Central - PubMed

Affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.

ABSTRACT
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

No MeSH data available.


Related in: MedlinePlus